Chronic alcoholism commonly results in deficiency of the vitamin folic acid. Studies in humans have shown that the folate deficiency results from a folate-depleting effect of ethanol as well from the poor diet of the alcoholic. Studies in rats have suggested that ethanol consumption produces an excess urinary lose of folate and that this may be a major mechanism for the folate depletion. The mechanism for the effect of ethanol on folate excretion is not known, although current data suggest an effect on the kidney's handling of folate. Studies in the present application are designed to propagate a cell culture model system from the human kidney proximal tubule. Cells will be grown to confluency on filter chambers that allow for separate manipulation of the media on each side of the cell layer. As such, the transfer of folate from the apical or brush border side through the cell layer to the basolateral side will be measured as a model of folate reabsorption. The first step in the process, binding to the apical membrane will be characterized by incubation of cells at 4'C with varying concentrations of the physiological substrate, 5-CH3-H4PteGlu. The mechanism for the transport of folate across the membrane will be assessed by incubation of the cells with substrate under the influence of luminal factors such as pH, extracellular ion content and competing substrates. Characterization of the specific folate bound to the membrane, of that transported and metabolized in the cell layer, and of that transferred through the layer into the basolateral chamber will be accomplished by high pressure liquid chromatography, Preincubation of cells with a covalent blocker of brush border binding protein activity followed by incubation with folate substrate will assess the role of binding in the transport of folate by the proximal tubule. cells will be adapted to grow in folate-deficient media. Subsequent transport studies will examine whether the cells increase the uptake of folate to overcome the deficiency. These pilot studies of folate transport by the proximal tubule cell will provide the basis for further studies of the mechanism for the effect of ethanol on folate handling by the kidney.